Atractylon inhibits the tumorigenesis of glioblastoma through SIRT3 signaling

Abstract

Glioblastoma (GBM) is the most common primary malignant brain tumor. Although there are various treatments for glioblastoma including surgery, radiotherapy, systemic therapy (chemotherapy and targeted therapy) and supportive therapy, the overall prognosis remains poor and the long-term survival rate is very low. Atractylon, a bioactive compound extracted from the Chinese herb Atractylodes lancea (Thunb.) DC. or Atractylodes chinensis (DC.) Koidz., has been reported to induce apoptosis and suppress metastasis in hepatic cancer cells. However, the roles and mechanisms of atractylon in GBM cells remain unknown. In the present study, we aimed to evaluate the effects of atractylon on the anti-tumorigenesis properties of GBM. Firstly, results of CCK8, colony formation, cell proliferation, and flow cytometry assays showed that atractylon inhibited the proliferation of GBM cells by arresting cells at the G1 phase of cell cycle. In addition, atractylon suppressed the migration and induced apoptosis of GBM cells. Mechanistically, atractylon treatment caused a significant up-regulation of sirtuin 3 (SIRT3, a tumor suppressor) mRNA and protein in GBM cells. Furthermore, inhibition of SIRT3 by the selective SIRT3 inhibitor 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP) partially restored the anti-proliferation and migration effects of atractylon in GBM cells. Finally, atractylon treatment also inhibited the in vivo growth of GBM cells in xenograft models through SIRT3 activation. Taken together, these results reveal a previously unknown role of atractylon in inhibiting GBM in vitro and in vivo through up-regulating SIRT3, which suggests novel strategies for the treatment of GBM.

Keywords: Atractylon; SIRT3; apoptosis; glioblastoma; migration; proliferation; tumorigenesis.

Figure 5

Atractylon inhibited C6 cell proliferation and migration via activating SIRT3 signaling in vitro. (A) The viability of C6 cells treated with different 3-TYP concentrations was determined by CCK8 assays (n=5 per group). (B) The viability of C6 cells treated with atractylon and 3-TYP was determined by CCK8 assays (n=5 per group). (C) Colony formation assays detecting the proliferation of C6 cells respectively treated with 100 μM atractylon for 24 h, 1 μM 3-TYP for 24 h, and 100 μM atractylon and 1 μM 3-TYP for 1 week. Scale bar, 700 mm. (D) Quantitative analysis of the surviving colonies as shown in (C) (n=3 per group). (E) Immunostaining of PH3 (red) in C6 cells with different treatment. Scale bar, 50 μm. (F) Quantitative analysis of the percentage of PH3 positive cells over total C6 cells in one filed as shown in (E) (n=6 per group). (G) Representative images from three independent experiments of C6 cells treated with vehicle control, 100 μM atractylon, 1 μM 3-TYP, or 100 μM atractylon and 1 μM 3-TYP in wound healing assays. Phase-contrast images were acquired at 0, 24, and 48 h after scratching. Scale bar, 900 μm. (H) Quantitative analysis of the relative wound healing area as shown in (G). (I) The expression of SIRT3, P53, and cleaved caspase-3 in C6 cells treated with vehicle control, or 100 μM atractylon, 1 μM 3-TYP, 100 μM atractylon and 1 μM 3-TYP was detected by western blot. (J-L) Quantitative analysis of the relative levels of cleaved caspase-3 (J), SIRT3 (K) and p53 (L) as shown in (I) (n=3 per group). Data were mean ± SEM. *P < 0.05, **P < 0.01.

Figure 1

Atractylon decreased the viability and inhibited the proliferation of GBM cells in vitro. (A) The viability of C6 cells treated with different atractylon concentrations as determined by CCK8 assays (n=5 per group). (B) The cell viability of DBTRG cells treated with different atractylon concentrations as determined by CCK8 assays (n=5 per group). (C) Representative images of C6 and DBTRG cells treated with 100 μM atractylon for 7 d in colony formation assays. Scale bar, 10 mm. (D) Quantitative analysis of the CV-stained colonies dissolved in 33% acetic acid and measured by absorbance at 570 nm as shown in (C) (n=4 per group). (E) Immunostaining of PH3 (green) in C6 and DBTRG cells treated with 100 μM atractylon for 24 h. Scale bar, 50 μm. (F) Quantitative analysis of the percentages of PH3-positive cells over total C6 and DBTRG cells in one field as shown in (E) (n=6 per group). Data were mean ± SEM. *P < 0.05, **P < 0.01.

Figure 2

Atractylon caused G1 cell cycle arrest and induced apoptosis in GBM cells. (A) Flow cytometric analysis of cell cycle distribution of C6 cells treated with 100 μM atractylon for 24 h (n=4 per group). (B) Quantitative analysis of the percentage of 2n-phase C6 cells as shown in (A). (C) Flow cytometric analysis of annexin V/FITC/PI stained control and treated (100 μM atractylon for 24 h) C6 cells. (D) Quantitative analysis of the apoptosis rate of C6 cells as shown in (C) (n=4 per group). (E) Representative image of PI staining in C6 cells after treatment with vehicle control or 100 μM atractylon for 24 h. Scale bar, 200 μm. (F) Quantitative analysis of the percentage of PI⁺ cells in total cells in one field as shown in (E) (n=4 per group). (G) The expression of P53, cleaved caspase-3, and cyclin D1 in treated (100 μM atractylon for 24 h) C6 cells was detected by western blot. (H-J) Quantitative analysis of P53 (H), cleaved caspase-3 (I), and cyclin D1 (J) levels as shown in (G) (n=3 per group). Data were mean ± SEM. *P < 0.05, **P < 0.01.

Figure 3

Atractylon inhibited the migration of GBM cells. (A) Representative images from three independent experiments of C6 and DBTRG cells treated with 100 μM atractylon in wound healing assays. Phase-contrast images were acquired at 0, 24 and 48 h after scratching. (B, C) Quantitative analysis of the relative wound healing area (normalized to 0 h) (n=20 per group). Scale bar, 900 μm. (D) Representative images of C6 and DBTRG cells treated with 100 μM atractylon for 24 h in transwell migration assays. Scale bar, 100 μm. (E, F) Quantitative analysis of the number of migrated C6 cells (E) or DBTRG cells (F) as shown in (D, n=6 per group). Data were mean ± SEM. *P < 0.05, **P < 0.01.

Figure 4

Atractylon treatment upregulated SIRT3 expression in C6 cells. (A, B) Survival analysis of SIRT3 expression in all primary (A) and recurrent (B) grade glioma. (C) 3D docking mode between atractylon and SIRT3 based on Discovery Studio assimilation and active site-amino acids (Leu 363, Lys, 219, and Val 360). (D) 2D diagram of the predicted interactions between atractylon and SIRT3; backbone of the protein is colored in grey. Ligand-protein interactions are colored depending on their type: alkyl is colored in purple, conventional hydrogen bonds are colored in brown. (E) The expression of SIRT3 and Lamin B1 in C6 cells treated with 100 μM atractylon for 24 h was detected by western blot. (F, G) Quantitative analysis of the relative SIRT3 (G) and Lamin B1 (F) levels as shown in (E) (n=3 per group). (H) Immunostaining of SIRT3 (red) in C6 cells treated with 100 μM atractylon for 24 h. (I) Quantitative analysis of the relative average fluorescence intensity of SIRT3 in single C6 cell as shown in (H) (n=6 per group). Scale bar, 25 μm. (J) qPCR analysis results of SIRT3 mRNA levels in C6 cells treated with atractylon at 100 μM for 24 h (n=4 per group). Data were mean ± SEM. *P < 0.05, **P < 0.01.

Figure 6

Atractylon inhibited the tumorigenesis of GBMs by activating SIRT3 signaling in vivo. (A) Representative images of C6 xenograft tumors after treatment with vehicle control or 20 mg/kg atractylon for 23 d. (B) Tumor weight in each group (n=10 per group). (C) The expression of P53, cleaved caspase-3, and SIRT3 in xenograft tumor tissues after treatment with vehicle control or 20 mg/kg atractylon for 23 d was detected by western blot. (D-F) Quantification of the relative levels of p53 (D), cleaved caspase-3 (E), and SIRT3 (F) as shown in (C) (n=3 per group). (G) Immunostaining of PH3 (green) in tumor tissues from mice treated with vehicle control or 20 mg/kg atractylon for 23 d. Scale bar, 50 μm. Data were mean ± SEM. *P < 0.05, **P < 0.01.

Figure 7

A working model of atractylon action on GBM cells. Atractylon treatment activates the expression of SIRT3 in GBM cells, which may in turn inhibit typical GBM tumorigenic characteristics, including blocking cell proliferation, suppressing cell migration, and inducing the apoptosis of GBM cells both in vitro and in vivo.